Actin, a contractile protein, is represented by multigene families in warm blooded vertebrate genomes. Actin is expressed in myogenic tissues such as skeletal, cardiac and smooth muscle. Some very low level expression of actin has been noted in non-myogenic tissue. The .alpha.-skeletal actin gene is a single copy gene, highly conserved during vertebrate evolution, and is expressed exclusively at high levels in adult skeletal muscle. The skeletal .alpha.-actin gene has been characterized in chicken (Fornwald et al., Nucleic Acids Res., Vol. 10, pp.3861-3876 (1982)), rat (Zak et al., Nature, Vol. 298, pp.857-859 (1982)), mouse (Hu et al., Mol. Cell. Biol., Vol. 6, pp.15-25 (1986)) and human (Minty and Kedes, Mol. Cell. Biol., Vol. 6, pp.2125-2136 (1986)) genomes at the level of nucleic acid sequencing.
Genes that have been characterized and sequenced such as the chicken skeletal .alpha.-actin gene can be mapped to determine the region of the gene that is not expressed and the region of the gene that is expressed to code the .alpha.-actin protein. Parts of the unexpressed portion of the gene determines gene expression, that is when and how the expressed gene is copied in the cell. Part of the process includes mRNA transcription which is necessary for gene expression.
Usually specific regulatory sequences or elements are embedded adjacent to or within protein coding regions of DNA. These elements, located adjacent to the gene, are termed cis-acting elements. These signals are recognized by other diffusable biomolecules in trans to potentiate the transcriptional activity. These biomolecules are termed trans-acting factors. The presence of trans-acting factors and cis-acting sequences, and their appropriate association has been shown to determine the expression activity of a gene.
Cis-acting elements can be further divided into three parts, the proximal promoter, the distal promoter and the enhancer/silencer sequences. The proximal promoter usually refers to a DNA fragment of about 50 bp to 100 bp in the 5'-flanking DNA upstream of the cap site or the RNA transcriptional initiation site. The proximal promoter can drive transcription at a basal level activity. The distal promoter is a sequence further 5' to the proximal promoter. Its major function is to modulate the transcriptional activity in a specific pattern, e.g., metal induction (Anderson et al., Mol. Cell. Biol., Vol. 7, pp.3579-3581 (1987)), heat shock induction (e.g. Wu, Nature, Vol. 309, pp.229-241 (1984)) or cAMP induction (Jameson et al., Mol. Cell. Biol., Vol 7, pp.3032-3040 (1987)). The enhancer or silencer is a sequence, which can be located at any distance from the gene, and function to increase or decrease the gene expression in an orientation-independent manner. The initiation of transcription is then determined by the factors recognizing these sequences. The enhancer binding factors and the distal promoter binding factors may interact with each other and influence the events at the proximal promoter by either promoting the formation of a transcription complex or enhancing the initiation of the RNA polymerase II on the DNA template.
Some gene expression such as .alpha.-actin is tissue specific such that the levels of the protein expressed is different in myogenic and non-myogenic tissue. Transient transfection experiments demonstrated that 411 nucleotides of DNA flanking the 5' end of the skeletal .alpha.-actin gene appeared to be responsible for its tissue restricted (not inducible in primary brain cell cultures, and COS-7) and developmentally appropriate transcription of the bacterial reporter gene chloramphenicol acetyltransferase (CAT) in chicken primary myoblasts. Grichnik et al., "Tissue restricted and stage specific transcription is maintained with 411 nucleotide flanking 5' end of the chicken .alpha.-skeletal actin gene.", Nucleic Acids Res., Vol. 14, pp. 1683-1701 (1986). It was also shown by S1mapping that the fusion gene CAT mRNA transcripts are under the transcriptional control of the .alpha.-actin 5'-flanking region and are accurately initiated at a surrogate start site 32 bp upstream of the CAT AUG translation initiation site.
The cis-acting regions of the chicken skeletal .alpha.-actin gene have been examined and located to some extent in the 5'-flanking region. Transgenic mice with the integrated skeletal .alpha.-actin promoter with the cis-acting regions showed preferential expression of the CAT gene in myogenic tissue. Schwartz et al., "Identification of Cis-Acting Regulatory Elements of the Chicken Skeletal .alpha.-Actin Gene Promoter," Cellular and Molecular Biology of Muscle Development, pp. 653-667, Alan R. Liss, Inc. (1989) and Bergsma et al., "Delimitation and Characterization of cis-acting DNA sequences Required for the Regulated Expression and Transcriptional Control of the Chicken .alpha.-Actin Gene," Molecular and Cellular Biology, Vol. 6, No. 7, pp. 2462-2475 (July 1986). Both of the foregoing articles are incorporated by reference herein.
The promoter was determined to have a 202 base pair 5'-flanking region which includes several cis-acting elements. The first regulatory site 5' to the cap site is the ATAAAA box between -29 bp and -24 bp which appears to be analogous to the TATA box which is involved with accurate initiation of most RNA polymerase II transcribed genes. Mutations of the gene made by nucleotide substitutions were used to identify the proximal promoter and distal promoter. The proximal promoter also termed the downstream promoter, is -94 bp to -74 bp 5' to the cap site and a CCAAT Box Associated Repeats (CBARs). Similar CBAR sequences have been found in many muscle-specific genes. The distal promoter is an upstream CBAR between -138 bp and -123 bp.
Mutations in the downstream CBAR regions caused complete inhibition of the promoter region in myogenic and non-myogenic cells. Mutation in the upstream CBAR reduced levels to 5% or less in myogenic cells. (Schwartz et al. 1989). Most of the mutations studied were in the cis-acting regions. One mutation at -154 bp to -149 bp increased tissue expression three-fold in non-myogenic tissue (fibroblasts) was more than 10 base pairs from the upstream CBAR. None of the mutated promoter regions contained nucleotide substitutions in more than one site.